Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with an array of changing environmental conditions to successfully persist, proliferate and be transmitted between hosts. The bacterial outer surface represents the primary site for interactions with the host. The array of B. burgdorferi outer surface proteins (Osps) has been shown to vary with the infectious cycle. It is likely that these different Osps endow the spirochete with distinct properties relevant to the disparate environments in which it must survive. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation in B. burgdorferi and their roles in the infectious cycle. We have previously demonstrated that temperature represents an important environmental variable, and that synthesis of a number of Osps is increased after a rise in culture temperature. Protein levels parallel transcript levels, consistent with regulation at the level of gene expression. The complete genomic sequence of B. burgdorferi identified homologs of three transcription factors, sigma 70, sigma 54, and RpoS. We are interested in the roles of RpoS and sigma 54 in the temperature induction and transcriptional regulation of osp genes. We have cloned the borrelial sigma 54 and rpoS genes and demonstrated partial complementation of a Shigella flexneri rpoS mutant with the B. burgdorferi rpoS gene. We have inactivated the B. burgdorferi rpoS gene and demonstrated that this mutant exhibits decreased osmotic resistance during stationary phase. A comparison of total bacterial proteins by two-dimensional gels revealed several differences between wild type and rpoS mutant spirochetes. Ongoing studies are aimed at the identification of these proteins. The genomic sequence of B. burgdorferi also identified a homolog of a transporter for the disaccharide chitobiose (cel). The degradation of chitin (a component of tick cuticle) to chitobiose could provide an important source of carbohydrate for B. burgdorferi in the tick. We have begun a genetic analysis of the cel genes to determine their role and function in B. burgdorferi. We have inactivated one of these genes, celB, and shown that this mutant grows as well as wild type B. burgdorferi in complex growth medium. Preliminary results indicate that growth of the celB mutant is impaired relative to wild type B. burgdorferi in medium lacking N-acetyl glucosamine (NAG), suggesting that uptake of chitobiose via the Cel transporter may fulfill the requirement for NAG. - Borrelia burgdorferi, spirochete, Lyme disease, molecular genetics, tick transmission, adaptive response